Enhanced protein phosphorylation is one of the intracellular events which invariably follows membrane receptor activation. Recently identified Protein Kinase C (PKC) is a unique enzyme which phosphorylates specific proteins, whose functions remain to be determined. PKC requires a phospholipid and a diacylglycerol (DAG) for maximal activity. DAG is the hydrolytic product of membrane polyphosphoinositide breakdown, which occurs following membrane receptor occupancy, and is one of the initial events in signal transduction. The release of DAG during PI turnover substantially stimulates PKC activity over that observed in quiescent cells. The known co-carcinogen or tumor promoter, TPA, specifically increases membrane PKC activity. Associated with this increased membrane PKC activity is a reduction of cytosolic PKC activity, which suggests that translocation of PKC from the cytosol to the membrane is an important intracellular event. Using AtT-20 cells that respond to a variety of membrane and intracellular activators by releasing POMC-derived peptides, we have explored the involvement of PKC in hormone secretion by monitoring the release of beta endorphin (BE), as well as membrane and cytosolic PKC activity. TPA treatment resulted in a dose-related increase in BE secretion and translocation of PKC from the cytosolic to the membrane fraction. A maximally-stimulating concentration of TPA enhanced membrane PKC activity 2-fold within 1 min and 5-fold within 3 min. Preincubation of AtT-20 cells with ethanol (0.1-0.6%) for 24 hours resulted in a dose-related 2-5 fold reduction in cytosolic PKC activity and a 50% reduction in membrane PKC activity at 0.4% ethanol. Ethanol pretreatment for 24 hours only marginally reduced the ability of a maximal stimulatory concentration of TPA to induce translocation of PKC.